Wang Di, Li Shaowu, Zhao Jingzhuang, Liu Hongbai, Lu Tongyan, Yin Jiasheng
Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, PR China.
Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, PR China.
Fish Shellfish Immunol. 2016 Sep;56:303-309. doi: 10.1016/j.fsi.2016.07.027. Epub 2016 Jul 21.
Hepcidin, an antimicrobial peptide, plays a crucial role in innate immune system of teleost fish. As a cysteine-rich peptide, hepcidin possesses a dual function including iron regulation and innate immunity. In the present study, a full-length hepcidin cDNA (HtHep) was cloned and characterized by RT-PCR and RACE techniques from taimen (Hucho taimen, Pallas), which is a type of rare, precious and cold-water fish species in China. The cDNA contains an open reading frame (ORF) of 267 bp encoding 88 amino acid (aa), with 170 bp located in the 5(') untranslated region (UTR) and 151 bp in the 3' UTR. The genomic sequences analysis showed that the HtHep gene consisted of three exons and two introns (with the length 94 and 251 bp, respectively). With a predicted molecular mass of 2881.4 Da and a theoretical pI of 8.53, the deduced amino acid encodes a signal peptide of 24 aa, prodomain of 39 aa and mature peptide of 25 aa. The signal peptidase (SA-VP) and the motif RX (K/R)R of propeptide convertase suggested the cleavage site of signal and mature peptide. Eight conserved cysteine residues were also identified and formed four disulfide bonds. Pair-wise alignments showed that HtHep clustered together with two fish species of Salmonidae family (Salmo salar and Oncorhynchus mykiss) in HAMP1 branch. Quantitative RT-PCR analysis indicated that the mRNA levels of HtHep were detected in a wide range of tissues and the highest level was detected in the liver. Its expression was also detected early during embryonic stage and could be up-regulated in the liver when challenged with pathogenic bacteria (Yersinia ruckeri). The recombinant HtHep (rHtHep) had antimicrobial activity against both gram-positive (Micrococcus lysodeikticus and Staphylococcus aureus) and gram-negative bacteria (Escherichia coli). Our results suggested that HtHep might be involved in the innate immune defense against bacterial pathogens in taimen.
抗菌肽铁调素在硬骨鱼的先天免疫系统中发挥着关键作用。作为一种富含半胱氨酸的肽，铁调素具有包括铁调节和先天免疫在内的双重功能。在本研究中，通过RT-PCR和RACE技术从哲罗鲑（Hucho taimen，Pallas）中克隆并鉴定了铁调素全长cDNA（HtHep），哲罗鲑是中国一种珍稀的冷水鱼类。该cDNA包含一个267 bp的开放阅读框（ORF），编码88个氨基酸（aa），5'非翻译区（UTR）有170 bp，3'UTR有151 bp。基因组序列分析表明，HtHep基因由三个外显子和两个内含子组成（长度分别为94和251 bp）。推导的氨基酸预测分子量为2881.4 Da，理论pI为8.53，编码一个24 aa的信号肽、一个39 aa的前结构域和一个25 aa的成熟肽。信号肽酶（SA-VP）和前体肽转化酶的基序RX（K/R）R表明了信号肽和成熟肽的切割位点。还鉴定出八个保守的半胱氨酸残基，并形成四个二硫键。成对序列比对显示，HtHep在HAMP1分支中与鲑科的两种鱼类（大西洋鲑和虹鳟）聚集在一起。定量RT-PCR分析表明，在多种组织中均检测到HtHep的mRNA水平，其中肝脏中的水平最高。在胚胎发育早期也检测到了它的表达，当受到病原菌（鲁氏耶尔森菌）攻击时，肝脏中的表达会上调。重组HtHep（rHtHep）对革兰氏阳性菌（溶壁微球菌和金黄色葡萄球菌）和革兰氏阴性菌（大肠杆菌）均具有抗菌活性。我们的结果表明，HtHep可能参与了哲罗鲑对细菌病原体的先天免疫防御。
